The present invention relates to recombinant DNA which encodes the SnaBI restriction endonuclease as well as SnaBI methylase, and to the production of SnaBI restriction endonuclease from the recombinant DNA.
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria. When they are purified away from other bacterial components, these restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the `recognition sequence`) along the DNA molecule. Once bound, they cleave the molecule within, to one side of, or to both sides of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred restriction endonucleases with unique specificities have been identified among the thousands of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res. 24:223-235 (1996)).
Restriction endonucleases are named according to the bacteria from which they are derived. Thus, the three different restriction endonucleases synthesized by Deinococcus radiophilus, for example, are named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5'TTTAAA 3', 5'PuGGNCCPy3' and 5'CACNNNGTG3' respectively. Likewise, the single restriction endonuclease synthesized by Escherichia coli RY13 is termed EcoRI. It recognizes the sequence 5'GAATTC3'.
Restriction endonucleases generally occur together with a second enzyme, the modification methylase. Together, the two enzymes form bacterial restriction-modification (R-M) system. Methylases are complementary to restriction endonucleases and they provide a way for bacteria to protect their own DNA from self-digestion. Modification methylases recognize and bind to the same recognition sequence as the restriction endonuclease they accompany, but instead of cleaving the DNA, they chemically modify one of the nucleotides within the sequence by the addition of a methyl group to form 5-methylcytosine, N.sup.4 -methylcytosine, or N.sup.6 -methyladenine. Following this methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by virtue of the activity of its modification methylase(s), and therefore it is completely insensitive to the presence of the endogenous restriction endonuclease(s). It is only unmodified, that is to say foreign, DNA that is sensitive to restriction endonuclease recognition and cleavage.
With the advent of recombinant DNA technology, it is possible to clone genes and overproduce the enzymes they encode in large quantities. The key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method to identify such clones within complex `libraries`, i.e. populations of clones derived by `shotgun` procedures, when they occur at frequencies as low as 10.sup.-3 to 10.sup.-4. Preferably, the method should be selective, such that the unwanted majority of clones are destroyed while the desirable rare clones survive.
Restriction-modification systems are being cloned with increasing frequency. The first cloned systems used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178:717-719 (1980); HhaII: Mann et al., Gene 3:97-112 (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78:1503-1507 (1981)). Since the presence of restriction-modification systems in bacteria enable them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from libraries that have been exposed to phages. This method has been found, however, to have only limited value. Specifically, it has been found that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning plasmids (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12:3659-3676 (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406 (1983); Theriault and Roy, Gene 19:355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164:501-509 (1985)).
A third approach to clone R-M systems is by selection for an active methylase gene (U.S. Pat. No. 5,200,333 and BsuRI: Kiss et al., Nucl. Acids. Res. 13:6403-6421 (1985)). Since R and M genes are usually closely linked, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10:219-225 (1980); BcnI: Janulaitis et al., Gene 20:197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21:111-119 (1983); and MspI: Walder et al., J. Biol. Chem. 258:1235-124 (1983)).
Another approach to clone R-M Systems in E. coli makes use of the fact that certain modification genes, when cloned into a new host and adequately expressed, enable the host to tolerate the presence of a different restriction gene. (Wilson et al; U.S. Pat. No.: 5,246,845 (1993)).
A more recent method, the "endo-blue method", has been described for direct cloning of restriction endonuclease genes in E. coli based on the indicator strain of E. coli containing the dinD::lacZ fusion (Fomenkov et al., U.S. Pat. No. 5,498,535; Fomenkov et al., Nucl. Acids Res. 22:2399-2403 (1994)). This method utilizes the E. coli SOS response following DNA damages caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535).
Because purified restriction endonucleases, and to a lesser extent, modification methylases, are useful tools for analyzing and rearranging DNA molecules in the laboratory, there is a strong commercial incentive to engineer bacterial strains that produce these enzymes in large quantities. Such overexpression strains increase the enzyme yield and simplify the task of enzyme purification.